Zhu Chuiyu, Li Junjie, Yu Haibo, Xu Kexin, He Xiaomei, Liu Zhihao, Chen Juan
Department of Infectious Diseases, Key Laboratory of Molecular Biology for Infectious Diseases (Ministry of Education), Institute for Viral Hepatitis, the Second Affiliated Hospital, Chongqing Medical University, Chongqing, PR China.
Key Laboratory of Clinical Laboratory Diagnostics (Chinese Ministry of Education), College of Laboratory Medicine, Chongqing Medical Laboratory Microfluidics and SPRi Engineering Research Center, Chongqing Medical University, Chongqing, 400016, PR China.
Anal Chim Acta. 2025 Oct 8;1370:344389. doi: 10.1016/j.aca.2025.344389. Epub 2025 Jul 5.
Hepatitis B virus (HBV) infection represents a major global public health challenge. The covalently closed circular DNA (cccDNA), which serves as the key reservoir for viral persistence, currently requires invasive liver biopsy for clinical monitoring. Recent studies have identified serum HBV pregenomic RNA (pgRNA) and its splicing variants emerging as potential noninvasive and reliable biomarkers for tracking disease progression and forecasting prognosis in chronic HBV infection. Precise detection of pgRNA is therefore essential for informed clinical decision-making and optimized patient management.
In this study, we developed a rapid, accurate, and clinically applicable method for HBV RNA detection by integrating CRISPR/Cas13a with reverse transcription-multienzyme isothermal rapid amplification (RT-MIRA). This innovative platform enabled simultaneous amplification of two pgRNA targets within a single-tube RT-MIRA reaction, allowing dual-target detection via one-step amplification. The optimized system achieved efficient isothermal amplification with a detection sensitivity of 10 copies/mL for both total and spliced pgRNA and exhibited no cross-reactivity with other common clinical viruses. Validation using both RT-qPCR and the RT-MIRA-Cas13a assay on clinical samples from 48 HBV-infected patients demonstrated a positive prediction value of 97.2 % and a negative predictive value of 100 %. These results conclusively validate the assay's reliability for clinical application in detecting and quantifying both total pgRNA and its splicing variants.
This study presents a rapid and user-friendly HBV RNA detection platform capable of accurately identifying both total and spliced pgRNA. The RT-MIRA-Cas13a assay demonstrates excellent clinical potential for prognosis assessment and disease monitoring in HBV infection. Its robust performance and operational simplicity suggest strong suitability for point-of-care applications, potentially transforming chronic HBV monitoring through accessible nucleic acid testing.
乙型肝炎病毒（HBV）感染是一项重大的全球公共卫生挑战。共价闭合环状DNA（cccDNA）是病毒持续存在的关键储存库，目前临床监测需要进行侵入性肝活检。最近的研究已确定血清HBV前基因组RNA（pgRNA）及其剪接变体是追踪慢性HBV感染疾病进展和预测预后的潜在非侵入性且可靠的生物标志物。因此，精确检测pgRNA对于明智的临床决策和优化患者管理至关重要。
在本研究中，我们通过将CRISPR/Cas13a与逆转录-多酶等温快速扩增（RT-MIRA）相结合，开发了一种快速、准确且适用于临床的HBV RNA检测方法。这个创新平台能够在单管RT-MIRA反应中同时扩增两个pgRNA靶点，通过一步扩增实现双靶点检测。优化后的系统实现了高效的等温扩增，对总pgRNA和剪接pgRNA的检测灵敏度均为10拷贝/毫升，并且与其他常见临床病毒无交叉反应。对48例HBV感染患者的临床样本同时使用RT-qPCR和RT-MIRA-Cas13a检测进行验证，结果显示阳性预测值为97.2%，阴性预测值为100%。这些结果最终验证了该检测方法在检测和定量总pgRNA及其剪接变体方面临床应用的可靠性。
本研究提出了一个快速且用户友好的HBV RNA检测平台，能够准确识别总pgRNA和剪接pgRNA。RT-MIRA-Cas13a检测在HBV感染的预后评估和疾病监测方面显示出优异的临床潜力。其强大的性能和操作简便性表明非常适合即时检测应用，有可能通过便捷的核酸检测改变慢性HBV监测方式。
